Study On The Resistance Mechanism Of Bt Toxin In Plodia Interpunctella (Hübner)

Plodia interpunctella,distributed all over the world,is one of the important pests in China.Most parts of our country have their harmful reports.Based on the knowledge of biochemistry and molecular biology,this project focuses on the problem of the resistance of reserve pests to transgenic Bt rice,P.interpunctella was chose as subject,we used the latest technologies such as Illumina sequencing,enzyme activity assays,RT-PCR,RNA interference,S-Poly(T)miRNA,to study the relationship between the midgut protective enzymes,the possible Bt toxin receptors-aminopeptidase N and cadherin genes with Bt toxin in P.Interpunctella,and further understand the toxicity mechanism of Bt rice to target pests.1.Transcriptome sequencing was performed for the susceptible and resistant strains of P.interpunctella and obtain 37246 Unigenes through De Novo assembling,There are 22211,14161,17755,15750,9348 and 10694 unigenes annotated to NR,NT,Swiss-Prot,KEGG,COG and GO,respectively.Totally 23310 Unigenes were annotated.The unigenes matched with Monarch butterfly was 67.6%.In order to further study the resistance mechanism of P.interpunctella to Bt toxin,we analyzed the genes which have significant differences in expression levels between the two strains.Finally,we found 15441 of the 34466 genes were up-regulated in the Bt stress strain and 18725 were down-regulated.The significantly differentially expressing genes between the two strains are 10224.Among them,9,55,10 and 4 genes encoding APN,Cadherin,ALP and Glycolipid,respectively.The genes encoding APN were significantly increased in the stress strain,while the other three receptor genes in the two strains have no significant difference.This indicated that the resistance mechanism of Bt toxin was closely related to APN.2.MicroRNAs(miRNA)are a group of small RNAs which participate in gene regulation and obtain more and more attention.However,there are few studies on miRNAs that regulate gene expression in P.Interpunctella were reported.The transcriptome of P.interpunctella libraries constructed from eggs,third-instar larvae,pupae and adults has been sequenced and analyzed to identify genes involved in development and insecticide resistance.In this study,we constructed and sequenced 12 small RNA libraries corresponding to three biological replicates of each of the four P.interpunctella developmental stages.In total,we obtained 173.2 million raw reads and identified 1906 known miRNAs and 588 novel miRNAs in P.interpunctella.A large number of the miRNAs were stage-specific.The target genes of the miRNAs were identified and annotated using GO Ontology and KEGG pathways.Expression trend analysis of the miRNAs and their target genes was also performed.The discovery of these small RNAs,particularly the miRNAs play important role in regulating the growth and development of P.Interpunctella,and also provides an important foundation for recognizing and defending against P.interpunctella from a a new perspective.3.We fed P.interpunctella 5th larvae with transgenic rice which have different concentrations and non-transgenic rice(control)for different time.Then the changes of three protective enzymes(SOD,CAT and POD)activities were studied.The results showed that,in the short term,SOD activity rose at first and then declined slowly as time increasing.The SOD activity in larvae had been significantly inhibited after fed with different Bt toxin concentrations for 12 days and 5 months.Its SOD activity decreased 38.75%after feeding on 100mg/g Bt transgenic rice for 5 months and had significant difference compared with the control.The POD activity in the larvae was always lower than control after feeding on 100mg/g transgenic Bt rice within 72h.However,the POD activity significantly increased compared to control after fed with different Bt toxin concentrations for 12 days and 5.months.After feeding on transgenic Bt rice for 48 h,the CAT activity became higher than control.Also,the CAT activity continued to rise with Bt toxin content increasing in the 12 days and 5 months experiments.The results suggested that the balance in activities of protective enzymes in the larvae of P.interpunctella was destroyed after a short treatment with Bt toxin,leading high levels of free radicals and resulting in an irreversible toxic effect on the larvae.However,exposed to transgenic Bt rice in long-term,its ability to resist Bt stressing had enhanced and formed a relatively complete defensive system.4.Using the RT-PCR combining with RACE technique,three full length sequence coding for two of putative APNs(PiAPN1,PiAPN2)and one of Cadherin(PiCad)were cloned.They contained a 2847bp,a 2844bp and a 5199 bp open reading frames(ORF)that encoded a 948-,a 947-and a 1732-amino acid proteins.The molecular weights(MW)of each protein sequence was 116.92,114,07 and 205.45 kDa,and the isoelectric points were calculated of 4.78,4.90 and 4.18,respectively.PiAPNl,PiAPN2 and PiCad contain a signal peptide and a different number of CR domains.APN-like and Cadherin-like proteins trom lepidopteran species downloaded and were used to constructed two phylogenetic frees through the Molecular Evolutionary Genetics Analysis(MEGA)software.The results showed that the amino acid sequences of PiAPN2 has the greatest similarity to the HaAPN(70%),while the PiAPN1 share a relative lower sequence homology with other genes.As for PiCad,it shares high sequence similarities in amino acid sequences with those known Cadherin genes of other lepidopteran species.5.The expression patterns of three genes above-mentioned were investigated by quantitative real-time PCR(qRT-PCR)in different developmental stages and upon Bt induction.The results showed that all three tested genes exhibited significantly high levels in larvae than other three stages and the Bt induction has a significant impact on the expression of these genes in P.interpunctella.RNA interference(RNAi)was employed through injection of the specific double-stranded RNA(dsRNA)for three target genes in the larvae of P.interpunctella.The expression of three genes were significantly down-regulated by RNAi and silencing of all three genes in P.interpunctella resulted in reduced susceptibilities to CrylAb toxin.These studies showed that the PiAPNl,PiAPN2 and PiCad we obtained were functionally associated with the Cry1Ab resistance in P.interpunctella.